Biofilms may interfere with membrane performance in at least three ways: (i) increase of the transmembrane pressure drop, (ii) increase of feed channel (feed-concentrate) pressure drop, and (iii) increase of transmembrane passage. Given the relevance of biofouling, it is surprising how few data exist about the hydraulic resistance of biofilms that may affect the transmembrane pressure drop and membrane passage. In this study, biofilms were generated in a lab scale cross flow microfiltration system at two fluxes (20 and 100Lm^-2h^-1) and constant cross flow (0.1ms^-1). As a nutrient source, acetate was added (1.0mgL^-1 acetate C) besides a control without nutrient supply. A microfiltration (MF) membrane was chosen because the MF membrane resistance is very low compared to the expected biofilm resistance and, thus, biofilm resistance can be determined accurately. Transmembrane pressure drop was monitored. As biofilm parameters, thickness, total cell number, TOC, and extracellular polymeric substances (EPS) were determined, it was demonstrated that no internal membrane fouling occurred and that the fouling layer actually consisted of a grown biofilm and was not a filter cake of accumulated bacterial cells. At 20Lm^-2h^-1 flux with a nutrient dosage of 1mgL^-1 acetate C, the resistance after 4 days reached a value of 6×10¹²m^-1. At 100Lm^-2h^-1 flux under the same conditions, the resistance was 5×10¹³m^-1. No correlation of biofilm resistance to biofilm thickness was found; Biofilms with similar thickness could have different resistance depending on the applied flux. The cell number in biofilms was between 4×10⁷ and 5×10⁸ cellscm^-2. At this number, bacterial cells make up less than a half percent of the overall biofilm volume and therefore did not hamper the water flow through the biofilm significantly. A flux of 100Lm^-2h^-1 with nutrient supply caused higher cell numbers, more biomass, and higher biofilm resistance than a flux of 20Lm^-2h^-1. However, the biofilm thickness after 4 days at a flux of 100Lm^-2h^-1 (97μm) was in the same order of magnitude as the thickness of a biofilm at a flux of 20Lm^-2h^-1 (114μm). An increase of flux caused an increased biofilm resistance while a decrease of flux caused a decreased resistance. The effect was reversible. It is suggested that the biofilm resistance is mainly attributed to EPS, probably due to the tortuosity ("hair-in-sink-effect") of the biopolymers to water molecules travelling across the biofilm. The data show clearly that biofilm resistance (6×10¹²m^-1) was high compared to the intrinsic resistance of the employed MF membrane (5×10¹¹m^-1). However, in nanofiltration (intrinsic membrane resistance ca. 2×10¹³m^-1) and reverse osmosis membranes (intrinsic resistance ca. 9×10¹³m^-1), the biofilm will not contribute significantly to the overall resistance.